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Zoledronic acid overcomes chemoresistance and immunosuppression of malignant mesothelioma.

Salaroglio IC, Campia I, Kopecka J, Gazzano E, Orecchia S, Ghigo D, Riganti C - Oncotarget (2015)

Bottom Line: We compared primary HMM samples with non-transformed mesothelial cells.By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes.Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Torino, Italy.

ABSTRACT
The human malignant mesothelioma (HMM) is characterized by a chemoresistant and immunosuppressive phenotype. An effective strategy to restore chemosensitivity and immune reactivity against HMM is lacking. We investigated whether the use of zoledronic acid is an effective chemo-immunosensitizing strategy. We compared primary HMM samples with non-transformed mesothelial cells. HMM cells had higher rate of cholesterol and isoprenoid synthesis, constitutive activation of Ras/extracellular signal-regulated kinase1/2 (ERK1/2)/hypoxia inducible factor-1α (HIF-1α) pathway and up-regulation of the drug efflux transporter P-glycoprotein (Pgp). By decreasing the isoprenoid supply, zoledronic acid down-regulated the Ras/ERK1/2/HIF-1α/Pgp axis and chemosensitized the HMM cells to Pgp substrates. The HMM cells also produced higher amounts of kynurenine, decreased the proliferation of T-lymphocytes and expanded the number of T-regulatory (Treg) cells. Kynurenine synthesis was due to the transcription of the indoleamine 1,2 dioxygenase (IDO) enzyme, consequent to the activation of the signal transducer and activator of transcription-3 (STAT3). By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

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Effects of Ras/ERK1/2 inhibition on STAT3 phosphorylation and IDO expression in mesothelioma cellsHMM samples (UPN: unknown patients number), treated with a TetON vector containing a shRNA for RAS (sh) or with the ERK1/2 inhibitor PD98059 (10 μmol/L for 24 h, PD), were cultured in medium without (−) or with (+) 1 μg/mL doxycycline (doxy). After 24 h, the following investigations were performed. (a) The expression of Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2 was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (b) The ERK activity was measured in cell lysates using a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (doxy -, PD -) cells: *p < 0.001. (c) The expression of phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3 was assessed in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (d) The relative expression levels of IDO mRNA were measured by qRT-PCR (open bars, mRNA); the kynurenine level in cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (solid bars, act). Data are presented as means ± SD (n = 4). Vs untreated (doxy -, PD -) cells: *p < 0.005.
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Figure 5: Effects of Ras/ERK1/2 inhibition on STAT3 phosphorylation and IDO expression in mesothelioma cellsHMM samples (UPN: unknown patients number), treated with a TetON vector containing a shRNA for RAS (sh) or with the ERK1/2 inhibitor PD98059 (10 μmol/L for 24 h, PD), were cultured in medium without (−) or with (+) 1 μg/mL doxycycline (doxy). After 24 h, the following investigations were performed. (a) The expression of Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2 was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (b) The ERK activity was measured in cell lysates using a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (doxy -, PD -) cells: *p < 0.001. (c) The expression of phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3 was assessed in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (d) The relative expression levels of IDO mRNA were measured by qRT-PCR (open bars, mRNA); the kynurenine level in cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (solid bars, act). Data are presented as means ± SD (n = 4). Vs untreated (doxy -, PD -) cells: *p < 0.005.

Mentions: Since Ras/ERK1/2 axis is involved in the serine phosphorylation of STAT3 [33, 34], we next investigated whether the zoledronic acid's effect was mediated by the down-regulation of Ras and ERK1/2 activity. To this aim, we produced two HMM clones (one epithelioid and one sarcomatous) stably and inducibly silenced for Ras: both clones showed decreased expression of Ras and phosphorylation of ERK1/2 (Figure 5a). The same clones were also incubated with the ERK1/2 inhibitor PD98059, used as second tool to block the Ras/ERK1/2 pathway. Compared to parental cells, both Ras-silenced and PD98059-treated cells showed lower ERK activity (Figure 5b), reduced levels of phospho(Ser727)-STAT3 without changes in phospho(Tyr705)-STAT3 or total STAT3 (Figure 5c), lower IDO activity and mRNA (Figure 5d), reproducing the situation observed in HMM cells treated with zoledronic acid.


Zoledronic acid overcomes chemoresistance and immunosuppression of malignant mesothelioma.

Salaroglio IC, Campia I, Kopecka J, Gazzano E, Orecchia S, Ghigo D, Riganti C - Oncotarget (2015)

Effects of Ras/ERK1/2 inhibition on STAT3 phosphorylation and IDO expression in mesothelioma cellsHMM samples (UPN: unknown patients number), treated with a TetON vector containing a shRNA for RAS (sh) or with the ERK1/2 inhibitor PD98059 (10 μmol/L for 24 h, PD), were cultured in medium without (−) or with (+) 1 μg/mL doxycycline (doxy). After 24 h, the following investigations were performed. (a) The expression of Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2 was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (b) The ERK activity was measured in cell lysates using a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (doxy -, PD -) cells: *p < 0.001. (c) The expression of phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3 was assessed in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (d) The relative expression levels of IDO mRNA were measured by qRT-PCR (open bars, mRNA); the kynurenine level in cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (solid bars, act). Data are presented as means ± SD (n = 4). Vs untreated (doxy -, PD -) cells: *p < 0.005.
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Figure 5: Effects of Ras/ERK1/2 inhibition on STAT3 phosphorylation and IDO expression in mesothelioma cellsHMM samples (UPN: unknown patients number), treated with a TetON vector containing a shRNA for RAS (sh) or with the ERK1/2 inhibitor PD98059 (10 μmol/L for 24 h, PD), were cultured in medium without (−) or with (+) 1 μg/mL doxycycline (doxy). After 24 h, the following investigations were performed. (a) The expression of Ras, phospho(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2, total ERK1/2 was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (b) The ERK activity was measured in cell lysates using a specific ELISA kit. Data are presented as means ± SD (n = 3). Vs untreated (doxy -, PD -) cells: *p < 0.001. (c) The expression of phospho(Tyr705)-STAT3, phospho(Ser727)-STAT3, total STAT3 was assessed in whole cell lysates by Western blotting. The β-tubulin expression was used as control of equal protein loading. The figure is representative of 3 experiments with similar results. (d) The relative expression levels of IDO mRNA were measured by qRT-PCR (open bars, mRNA); the kynurenine level in cell culture supernatant, taken as an index of IDO enzymatic activity, was measured fluorimetrically (solid bars, act). Data are presented as means ± SD (n = 4). Vs untreated (doxy -, PD -) cells: *p < 0.005.
Mentions: Since Ras/ERK1/2 axis is involved in the serine phosphorylation of STAT3 [33, 34], we next investigated whether the zoledronic acid's effect was mediated by the down-regulation of Ras and ERK1/2 activity. To this aim, we produced two HMM clones (one epithelioid and one sarcomatous) stably and inducibly silenced for Ras: both clones showed decreased expression of Ras and phosphorylation of ERK1/2 (Figure 5a). The same clones were also incubated with the ERK1/2 inhibitor PD98059, used as second tool to block the Ras/ERK1/2 pathway. Compared to parental cells, both Ras-silenced and PD98059-treated cells showed lower ERK activity (Figure 5b), reduced levels of phospho(Ser727)-STAT3 without changes in phospho(Tyr705)-STAT3 or total STAT3 (Figure 5c), lower IDO activity and mRNA (Figure 5d), reproducing the situation observed in HMM cells treated with zoledronic acid.

Bottom Line: We compared primary HMM samples with non-transformed mesothelial cells.By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes.Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Torino, Italy.

ABSTRACT
The human malignant mesothelioma (HMM) is characterized by a chemoresistant and immunosuppressive phenotype. An effective strategy to restore chemosensitivity and immune reactivity against HMM is lacking. We investigated whether the use of zoledronic acid is an effective chemo-immunosensitizing strategy. We compared primary HMM samples with non-transformed mesothelial cells. HMM cells had higher rate of cholesterol and isoprenoid synthesis, constitutive activation of Ras/extracellular signal-regulated kinase1/2 (ERK1/2)/hypoxia inducible factor-1α (HIF-1α) pathway and up-regulation of the drug efflux transporter P-glycoprotein (Pgp). By decreasing the isoprenoid supply, zoledronic acid down-regulated the Ras/ERK1/2/HIF-1α/Pgp axis and chemosensitized the HMM cells to Pgp substrates. The HMM cells also produced higher amounts of kynurenine, decreased the proliferation of T-lymphocytes and expanded the number of T-regulatory (Treg) cells. Kynurenine synthesis was due to the transcription of the indoleamine 1,2 dioxygenase (IDO) enzyme, consequent to the activation of the signal transducer and activator of transcription-3 (STAT3). By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

Show MeSH
Related in: MedlinePlus